大蒜新素抑制小鼠巨细胞病毒感染诱导调节性T细胞扩增的体外研究  

Role of allitridin in the blocking of murine cytomegalovirus induced regulatory T cells amplification in vitro

作　　者：李亚男[2] 王慧[1] 刘兴楼[1] 董永绥[1] 方峰[1] 

机构地区：[1]华中科技大学同济医学院附属同济医院儿科,武汉430030 [2]吉林大学第一医院儿内一科

出　　处：《中华医学杂志》2010年第28期1995-1998,共4页National Medical Journal of China

摘　　要：目的在细胞水平研究大蒜新素对小鼠巨细胞病毒（MCMV）感染鼠胚肺成纤维细胞（MEF）诱导调节性T细胞（Treg）异常扩增的抑制作用。方法建立MEF和T淋巴细胞的共培养体系，采用MEF对大蒜新素的最大耐受浓度（MTC）处理MCMV感染的MEF3d后，再用实时定量PCR检测T细胞中叉头蛋白3（Foxp3）mRNA表达水平，流式细胞术检测效应性T细胞亚群杀伤性T细胞Ⅰ型（Tcl）、杀伤性T细胞Ⅱ型（Tc2）、辅助性T细胞Ⅰ型（Thl）和辅助性T细胞Ⅱ型（Th2）的百分比，双抗体夹心酶联免疫吸附法检测共培养体系中白细胞介素10（IL）-10和转化生长因子（TGF）-β的蛋白表达，标准蚀斑法检测共培养体系中的病毒负荷量，并与安慰剂组进行统计学分析比较。结果MEF对大蒜新素的MTC浓度为9．83μg／ml。MTC浓度的大蒜新素可部分拮抗MCMV诱导的Foxp3基因表达上调（87±5比114±8，P〈0．01）；分别上调Tc1、Tc2和Th1百分比至（12．42±1．23）％、（4．28±0．56）％、（13．25±0．68）％，与安慰剂组[（6．85±0．92）％、（2．34±0．42）％、（9．32±0．86）％]比较差异均有统计学意义（均P〈0．01）；使IL-10和TGF-β蛋白表达水平分别降至（29．98±3．15）pg／ml和（3．48±0．23）ng／ml，均显著低于安慰剂组水平[（38．21±4．02）pg／ml和（5．31±0．59）ng／ml，均P〈0．05]；同时将体系中的病毒负荷量由（6．79±0．39）降至（5．03±0．08）（均P〈0．01）。结论大蒜新素在体外可通过抑制Treg途径来增强抗病毒免疫。Objective To investigate the effect of allitridin on murine cytomegalovirus （MCMV） induced regulatory T cells （Treg） amplification in vitro. Methods A co-culture system of T cells and MCMV infected mouse embryo fibroblasts （MEF） was established. A maximum tolerance concentration （MTC） of allitridin was added into the co-culture system. After 3 days, the change of Foxp3 mRNA was measured by real-time PCR. And the percentages of Tc1（ CD3^＋ CD8^＋ IFN-γ ）, Tc2 （ CD3^ ＋ CD8^ ＋ IL-4 ^＋ ）, Thl （ CD3 ^＋ CD8^ - IFN-γ^＋ ） and Th2 （ CD3^＋ CD8^ - IL-4^＋ ） were analyzed by flow eytometry. The production of IL-10 and TGF-β in supernatants was detected with double-antibody sandwich ELISA while the viral load of culture quantified by plaque assay. All results were compared with those of the placebo group. Results The MTC of allitridin was 9. 83 μg/ml in MEF. The treatment of 9. 83μg/ml allitridin could partly block the MCMV induction of Foxp3 mRNA expression [ （87 ± 5 ） vs （ 114 ± 8）, P 〈 0. 01 ]. The percentages of Te1, Tc2 and Thl significantly increased to the levels of （ 12.42 ± 1.23 ） %, （4. 28 ± 0. 56 ） % and （ 13.25 ± 0. 68 ） % respectively. They showed statistic differences with those of placebo controls [ （ 6. 85 ± 0. 92 ） % , （ 2. 34 ± 0. 42） % and （9. 32 ± 0. 86） % ; all P 〈 0. 01 ]. Meanwhile, the levels of IL-10 and TGF-β1 in supernatants also significantly decreased to （ 29. 98±3.15 ） pg/ml and （ 3.48 ± 0. 23 ） ng/ml by allitridin treatment as compared with placebo controls [ （ 38. 21 ± 4. 02 ） pg/ml and （ 5.31 ± 0. 59 ） ug/ml, all P 〈 0. 05]. In addition, the MCMV plaque assays showed that allitridin significantly suppressed the viral loads by one order of magnitude. Conclusion Mlitridin can partly retrieve MCMV-induced Treg expansion and Treg-mediated anti-MCMV immunosuppression so as to enhance the specific cellular immune responses against CMV.

关 键 词：鼠巨细胞病毒 T淋巴细胞 调节性 大蒜新素 

分 类 号：R28[医药卫生—中药学]